The Bacterial Diseases of the Bean 27 



Indol 'production. A good cloudy growth is produced in trytophane broth. 

 At the end of one and of four days, tests made for indol by the Ehrlich- 

 Bohme method show no trace of this substance. 



Nitrate reduction. A good cloudy growth is produced in nitrate broth but 

 nitrates are not reduced to nitrites. Tests were made at the end of 

 two, four, and eight days with sulfanilic acid and a-naphthalamine in 

 acetic acid. The brown pigment is formed in this medium. 



Hydrogen-sulfide production. No growth is obtained on Wilson's medium. 

 On lead-acetate agar a browning occurred along the stab, but this brown 

 coloration appeared to be the usual coloration produced by this organism 

 on most media. It is doubtful whether hydrogen sulfide is produced. 



Chromogenesis. The brown water-soluble pigment which is formed in 

 most media, owing to the action of the pathogene, is so striking in 

 appearance that it demands a certain amount of attention. It is a 

 phenomenon not commonly met with among the members of the bacterial 

 plant pathogenes, although it occurs not infrequently with species of 

 the genus Actinomyces. A brown pigment has been reported in litera- 

 ture as being produced by Phytomonas campestre and Phyt. hyacinthi, 

 but most descriptions of these organisms do not refer to it; and a sufficient 

 description has never been given to permit of determining whether or 

 not it is similar to the pigment formed in media by the action of the bean 

 pathogene. It is possible, too, that only certain strains of the two 

 bacteria referred to are capable of causing the formation of the brown 

 coloration of the media. The information is rather scanty. 



The brown coloration formed in cultures of certain species of Actino- 

 myces appears to be the same or a closely related product. With this 

 group of organisms the phenomenon is considered to be the result of an 

 enzymatic action on the amino acid, tyrosine, contained in the media, 

 and the production of melanin. It is probable that two enzymes produced 

 by the organism are implicated in the process. Waksman (1926) has 

 reviewed the literature on tyrosinase and has suggested the possible 

 chemical reactions which lesult in the brown substance formed. It will be 

 noted in the above cultural descriptions of the organism that the brown 

 color does not appear in synthetic media where tyrosine would be lacking. 

 Acting on this suggestion, Conn's (1921) synthetic medium for growing 

 species of Actinomyces was prepared. In one lot of this medium, tyrosine 

 replaced the sodium asparaginate. The organism grew fairly well on the 

 two simple media, and on the second day a purple-brown color appeared 

 in the tyrosine agar and deepened to a dark brown. No color appeared 

 in the sodium-asparaginate agar. For further confirmation, Phyt. phaseoli 

 was grown at the same time on the two media. After three weeks of 

 incubation a light brown color did appear on the tyrosine agar, but this 

 was considered to be due to a natural breaking-down of the amino acid 

 under these conditions. One-tenth of 1 per cent of tyrosine had been used 

 in this medium, which, no doubt, is an excessive amount. The difference 



